Cell are able to process an enormous amount of information about their environment and their internal status via highly regulated signaling pathways. Critical to this information transfer are small molecule second messengers, such as cAMP, Ca2+, and a recently emerging family of phosphorylated inositides. Among the many derivatives of the inositol phosphate messengers is an intriguing subgroup that possesses high energy diphosphate groups, namely the diphosphoinositol phosphates (or PP-IPs), which have recently been connected to several cellular functions, including vesicular trafficking and apoptosis. The PP-IPs have also been linked to abnormal physiological processes including diminished insulin secretion and cancer. Owing to their chemically complex nature, there exist significant techical challenges to uncovering inositol phosphate function. This proposal seeks to overcome these hurdles, using a combination of genetic and chemical methods, to elucidate the discret signaling functions of the PP-IPs. More specifically, the mentored phase of this award is aimed at interrogating PP-IP function genetically. Genome-wide quantitative genetic interaction mapping of the enzymes involved in PP-IP biosynthesis will reveal the functional connections of PP-IPs with all other proteins in S. cerevisiae (aim 1). Aim 2 will evaluate PP-IP function with respect to insulin secretion from pancreatic (3-cells. Here, the characterization of PP-IP function can help guide the development of novel antidiabetics. During the independent period of this award, aims 3, 4, and 5 will rely on the development of chemical probes to study PP-IP function. Through the development of a reagent for the affinity purification of diphosphorylated proteins, the impact of this novel post-translational modification will be evaluated (aim 3). PP-IP analogues that can be covalently linked to their protein binding partners will serve to map the preferred inositol phosphate binding sites (aim 4). Lastly, aim 5 will explore the metal binding properties of the PP-IPs, which will be exploited for the development of luminescent probes for in vivo imaging. By employing these tools in the cell models generated in aims 1 and 2, it will be possible to assess the physiological relevance of PP-IP signaling. Relevance: The diphosphoinositol phosphates are an important class of secondary messengers in the cell, yet they have received little consideration to date. Their function has clearly been linked to several diseases, including the emerging diabetes epidemic. Elucidation of their signaling function will help to develop novel and improved therapeutics against the development to type 2 diabetes.